Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus

ABSTRACT

Peptides are used to define epitopes that stimulate HLA-restricted cytotoxic T lymphocyte activity against hepatitis B virus antigens. The peptides are derived from regions of HBV polymerase, and are particularly useful in treating or preventing HBV infection, including methods for stimulating the immune response of chronically infected individuals to respond to HBV antigens.

GOVERNMENT SUPPORT

The U.S. Government may have certain rights in this invention pursuantto grants awarded by the National Institutes of Health.

RELATED APPLICATIONS

This application is a divisional of Ser. No. 08/416,950, filed Apr. 4,1995, which is a continuation of Ser. No. 08/100,870, filed Aug. 2,1993, abandoned, which is a continuation-in-part of Ser. No. 07/935,898,filed Aug. 26, 1992, abandoned which is a continuation-in-part of Ser.No. 07/749,540, filed Aug. 26, 1991, abandoned, each of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Cytotoxic T lymphocytes (CTLs) play an essential role in fighting cellsinfected with viruses, intracellular bacteria and parasites, and tumorcells. They do so by direct cytotoxicity and by providing specific andnonspecific help to other immunocytes such as macrophages, B cells, andother T cells. Infected cells or tumor cells process antigen throughintracellular events involving proteases. The processed antigen ispresented on the cellular surface in the form of peptides bound to HLAclass I molecules to T cell receptors on CTLs. MHC class I molecules canalso bind exogenous peptides and present them to CTLs withoutintracellular processing.

At the present time it is difficult to accurately predict from thesequence of an antigenic protein how the protein will be processed andwhich peptide portions will bind HLA class I molecules and be presentedto CTLs. Binding motifs have been predicted for some HLA class Imolecules based on sequence analysis of peptides eluted from thesemolecules (Falk et al., Nature 351:290 (1991)). Further, of the peptidesthat are processed and do bind to HLA class I, which ones will containCTL-recognizable epitopes is not yet predictable.

Hepatitis B Virus ("HBV") is a non-lytic virus which has currentlyinfected approximately 250 million people worldwide. HBV infection inadults typically leads to an acute disease in the majority of cases, andto a chronic disease state in a minority of patients. This ratio ofacute to chronic is reversed when the infection occurs close to the timeof birth. There is an increased incidence of hepatocellular carcinoma inchronic HBV infection. A small percentage of individuals who areinfected with HBV in adulthood develop fulminant hepatitis associatedwith a strong immune response with high lethality.

While there is no effective treatment for HBV infection, vaccines havebeen developed in recent years to prevent HBV infection. The vaccinesemploy either HBV surface antigen (HBsAg) purified from the plasma ofchronic HBV carriers, or HBsAg produced by recombinant DNA technology.Synthetic HBsAg peptide-based vaccines have also been proposed. See, forexample, U.S. Pat. Nos. 4,599,230 and 4,599,231. The anti-HBsAgvaccines, however, afford protection in only about 90% of immunizedindividuals. Those who are unimmunized, or immunized but unprotected,provide a significant reservoir of potential infection.

The contribution of CTLs to immunity to HBV antigens has been difficultto assess. Chisari et al. (Microbial Pathogen. 6:31 (1989)) havesuggested that liver cell injury may be mediated by an HLA-Class Irestricted, CD8⁺ cytotoxic T cell response to HBV encoded antigens.Class I major histocompatibility (MHC) -restricted cytotoxic Tlymphocyte responses have been identified for a variety of otherviruses, such as influenza. For example, Townsend et al., Cell 44:959(1986) reported that epitopes of an influenza virus nucleoproteinrecognized by cytotoxic T lymphocytes could be defined by syntheticpeptides. In attempting to define the cytotoxic T lymphocyte response toHBV, it has been shown that peripheral blood lymphocytes from patientswith acute and chronic HBV may be able to kill autologous hepatocytes invitro, but the specificity of the cytolytic activity, its HLArestriction elements, and cellular phenotype were not established. See,Mondelli et al., J. Immunol. 129:2773 (1982) and Mondelli et al., Clin.Exp. Immunol. 6:311 (1987). Moriyama et al., science 248:361-364 (1990),have reported that the HBV major envelope antigen is expressed at thehepatocyte surface in a form recognizable by envelope-specificantibodies and by MHC class I-restricted, CD8⁺ cytotoxic T lymphocytes.

As there is a large reservoir of individuals chronically infected withHBV, it would be desirable to stimulate the immune response of theseindividuals to respond to appropriate HBV antigens and thereby eliminatetheir infection. It would also be desirable to prevent the evolution toa chronic HBV infection in individuals suffering from an acute phaseinfection. Further, as the presently approved HBV vaccines do not elicitprotective immunity in about 10% of immunized individuals, it would bedesirable to elicit more effective immunity, such as by increasing ordiversifying the immunogenicity of the vaccines. Quite surprisingly, thepresent invention fulfills these and other related needs.

SUMMARY OF THE INVENTION

The present invention provides peptides which induce MHC class Irestricted cytotoxic T lymphocyte responses against HBV antigen. Thepeptides of interest are derived from the sequence of the HBV polymeraseprotein. In certain embodiments the CTL inducing peptide will have thesequence of HBpol61-69 (Seq. ID No. 1),Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val; HBpol 455-463 (SEQ ID NO 2),Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu; HBpol 773-782 (SEQ ID NO 3),Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val; HBpol803-811 (Seq. ID No. 4),Ser-Leu-Tyr-Ala-Asp-Ser-Pro-Ser-Val; or HBpol816-824 (Seq. ID No. 5),Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu, or will have a sequencesubstantially homologous to one of the foregoing sequences. The peptidecan be optionally flanked and/or modified at one or both of the N- andC-termini, as desired. Conservative substitutions, deletions andadditions may be made at non-critical residue positions within theselected peptide without substantially adversely affecting itsbiological activity.

In the various peptide embodiments it will be understood that thepeptides can be polymerized, each to itself to form larger homopolymers,or with different peptides to form heteropolymers. In some instancespeptides will be combined in a composition as an admixture and will notbe linked. The peptide can also be conjugated to a lipid-containingmolecules capable of enhancing a T lymphocyte response, or to adifferent peptide which induces a T-helper cell response, for example.

Compositions are provided which comprise a peptide of the inventionformulated with an additional peptide, a liposome, an adjuvant and/or apharmaceutically acceptable carrier. Thus, pharmaceutical compositionscan be used in methods of treating acute HBV infection, particularly inan effort to prevent the infection from progressing to a chronic orcarrier state. Methods for treating chronic HBV infection and HBVcarrier states are also provided, where the pharmaceutical compositionsare administered to infected individuals in amounts sufficient tostimulate immunogenically effective cytotoxic T cell responses againstHBpol epitopes. For treating these infections it may be particularlydesirable to combine the peptides which induce MHC class I restrictedcytotoxic T lymphocyte responses against HBV antigen with other peptidesor proteins that induce immune response to other HBV antigens, such asHBV envelope or core. To treat individuals with chronic or carrier stateinfections the compositions may be administered in repeated dosages overa prolonged period of time, as necessary, to resolve or substantiallymitigate the infection and/or shedding of virus.

Vaccine compositions for preventing HBV infection, particularly chronicHBV infection, are also provided. The vaccine compositions comprise animmunogenically effective amount of a HBV polymerase peptide mentionedabove which induces a MHC class I restricted cytotoxic T lymphocyteresponse, such as HLA-A2, and will typically further comprise anadjuvant, e.g., incomplete Freund's adjuvant or aluminum hydroxide. Toachieve enhanced protection against HBV, the vaccine can furthercomprise components which elicit a protective antibody response to otherHBV antigen, such as envelope (surface) antigen.

In yet other embodiments the invention relates to methods for diagnosis,where the peptides of the invention are used to determine the presenceof lymphocytes in an individual which are capable of a cytotoxic T cellresponse to HBV polymerase antigen. The absence of such cells determineswhether the individual of interest is susceptible to developing chronicHBV infection. Typically the lymphocytes are peripheral bloodlymphocytes and the individual of interest is suffering from an acuteHBV infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the CTL response to two polymerase peptides that containthe HLA-A2 motif in a patient using target cells pulsed with peptidethat match only at HLA-A2.

FIG. 2 shows the ability of several polymerase 803-811 peptide specificclones to recognize endogenously synthesized polymerase.

FIG. 3A shows that the CTL response to polymerase peptide 803-811 canrecognize cells pulsed with peptide and endogenously synthesizedpolymerase (Vpol), FIG. 3B indicates the CTL response to polymerasepeptide 61-69 only recognized cells pulsed with the 61-69 peptide.

FIG. 4A through D shows the aligned amino acid sequences of 20 clonedHBV polymerase proteins; line 158 is a consensus sequence where capitalletters represent 100% consensus, lower case letters represent >50%consensus, and "." is <50% consensus.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The present invention provides peptides derived from HBV polymeraseproteins for use in compositions and methods for the treatment,prevention and diagnosis of HBV infection. The peptides stimulate MHCHLA-class I restricted cytotoxic T lymphocyte responses against HBVinfected cells. The stimulated cytotoxic T lymphocytes are able to killthe infected cells or inhibit viral replication and thus interrupt orsubstantially prevent infection, including chronic HBV infection. Apeptide effective in eliciting a cytotoxic T cell response may also becombined with an immunogen capable of eliciting a T-helper response.

The peptides employed in the invention are derived from the sequence ofthe HBV polymerase protein (HBpol), particularly CTL epitopes withinHBpol₆₁₋₆₉, HBpol 455-463, HBpol 773-782, HBpol 803-811, and HBpol816-824, where the numbering is according to Galibert et al., supra.

By HBV cytotoxic T lymphocyte inducing "peptide" or "oligopeptide" ofthe present invention is meant a chain of at least four HBV amino acidsequence residues, preferably at least six, more preferably eight ornine, sometimes ten to twelve residues, and usually fewer than aboutfifty residues, more usually fewer than about thirty-five, andpreferably fewer than twenty-five, e.g., eight to seventeen amino acidresidues derived from an HBc sequence. It may be desirable to optimizepeptides of the invention to a length of eight to twelve amino acidresidues, commensurate in size with endogenously processed viralpeptides that are bound to MHC class I molecules on the cell surface.See generally, Schumacher et al., Nature 350:703-706 (1991); Van Bleeket al., Nature 348:213-216 (1990); Rotzschke et al., Nature 348:252-254(1990); and Falk et al., Nature 351:290-296 (1991), which areincorporated herein by reference. As set forth in more detail below,usually the peptides will have at least a majority of amino acids whichare homologous to a corresponding portion of contiguous residues of theHBV pol sequences herein, and contain a CTL-inducing epitope.

The peptides can be prepared "synthetically," as described hereinbelow,or by recombinant DNA technology. Although the peptide will preferablybe substantially free of other naturally occurring HBV proteins andfragments thereof, in some embodiments the peptides can be syntheticallyconjugated to native fragments or particles. The term peptide is usedinterchangeably with polypeptide in the present specification todesignate a series of amino acids connected one to the other by peptidebonds between the alpha-amino and alpha-carboxy groups of adjacent aminoacids. The polypeptides or peptides can be a variety of lengths, eitherin their neutral (uncharged) forms or in forms which are salts andeither free of modifications such as glycosylation, side chainoxidation, or phosphorylation or containing these modifications, subjectto the condition that the modification not destroy the biologicalactivity of the polypeptides as herein described.

Desirably, the peptide will be as small as possible while stillmaintaining substantially all of the biological activity of the largepeptide. By biological activity is meant the ability to bind anappropriate MHC molecule and induce a cytotoxic T lymphocyte responseagainst HBV antigen or antigen mimetic. By a cytotoxic T lymphocyteresponse is meant a CD8⁺ T lymphocyte response specific for an HBVantigen of interest, wherein CD8⁺, MHC class I-restricted T lymphocytesare activated. The activated T lymphocytes secrete lymphokines (e.g.,gamma interferon) liberate products (e.g., serine esterases) thatinhibit viral replication in infected autologous cells or transfectedcells, with or without cell killing.

The terms "homologous", "substantially homologous", and "substantialhomology" as used herein denote a sequence of amino acids having atleast 50% identity wherein one sequence is compared to a referencesequence of amino acids. The percentage of sequence identity or homologyis calculated by comparing one to another when aligned to correspondingportions of the reference sequence.

The peptides of the invention contain CTL-inducing epitopes derived fromvarious epitopic regions of the HBV polymerase protein. The peptides arefrom the region of HBpol₆₁₋₆₉ and include peptides derived from thosesequence regions which contain one or more CTL-inducing HLA classI-restricted epitopic site(s) of at least seven contiguous amino acids.A majority of the amino acids of the peptide will be identical orsubstantially homologous to the amino acids of the correspondingportions of the naturally occurring HBpol₆₁₋₆₉ sequence, whereHBpol₆₁₋₆₉ has the following sequence (for HBV subtype ayw):

           (HBpol.sub.61-69)  SEQ. ID No. 1!    Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val,

and

The peptide embodiments of this HBpol₆₁₋₆₉ region and the otherpolymerase peptide regions described herein can be optionally flankedand/or modified at one or both of the N- and C-termini, as desired, byamino acids from HBV sequences, including HBpol, amino acids added tofacilitate linking, other N- and C-terminal modifications, linked tocarriers, etc., as further described herein. The peptide HBpol61-69induces a cytotoxic T lymphocyte response which is mediated by at leastthe MHC class I molecule HLA-A2.

Other HBpol region peptides containing CTL epitopes of the inventioncomprises the peptide HBpol 455-463, and peptides derived fromHBpol455-463 which contain a CTL-inducing HLA class I-restrictedepitopic site(s) of at least seven contiguous amino acids. A majority ofthe amino acids of the peptide will be identical or substantiallyhomologous to the amino acids of the corresponding portions of thenaturally occurring HBpol455-463 sequence, where HBpol 455-463 has thesequence (for HBV subtype ayw):

       (HBpol 455-463)  SEQ ID NO 2!    Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu

wherein the selected peptide can be flanked and/or modified at one orboth termini as described herein. The peptide HBpol 455-463 induces acytotoxic T lymphocyte response which is mediated by at least the MHCclass I molecule HLA-A2. Other HBpol region peptides containing CTLepitopes of the invention comprises the peptide HBpol 773-782, andpeptides derived from HBpol773-782 which contain a CTL-inducing HLAclass I-restricted epitopic site(s) of at least seven contiguous aminoacids. A majority of the amino acids of the peptide will be identical orsubstantially homologous to the amino acids of the correspondingportions of the naturally occurring HBpol773-782 sequence, where HBpol773-782 has the sequence (for HBV subtype ayw):

         (HBpol 773-782)  SEQ ID NO 3!    Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val

wherein the selected peptide can be flanked and/or modified at one orboth termini as described herein. The peptide HBpol 773-782 induces acytotoxic T lymphocyte response which is mediated by at least the MHCclass I molecule HLA-A2. Other HBpol peptide embodiments of theinvention are prepared from the region of HBpol803-811. Peptides derivedfrom this region contain at least one CTL-inducing HLA classI-restricted epitopic site, and will typically be at least seven aminoacids, more usually nine, ten or eleven amino acids or more. A majorityof the amino acids of the peptide will be identical or substantiallyhomologous to the amino acids of the corresponding portions of thenaturally occurring HBpol803-811 sequence, where HBpol803-811 has thesequence (for HBV subtype ayw):

        (HBpol.sub.803-811)  SEQ. ID No. 4!    Ser-Leu-Tyr-Ala-Asp-Ser-Pro-Ser-Val,

wherein the selected peptide can be flanked and/or modified at one orboth termini as described herein. The peptide HBpol 803-811 induces acytotoxic T lymphocyte response which is mediated by at least the MHCclass I molecule HLA-A2.

Other HBpol peptide embodiments of the invention are prepared from theregion of HBpol816-824. Peptides derived from this region contain atleast one CTL-inducing HLA class I-restricted epitopic site, and willtypically be at least seven amino acids, more usually nine, ten oreleven amino acids or more. A majority of the amino acids of the peptidewill be identical or substantially homologous to the amino acids of thecorresponding portions of the naturally occurring HBpol816-824 sequence,where HBpol816-824 has the sequence (for HBV subtype ayw):

        (HBpol.sub.816-824)  SEQ. ID No. 5!    Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu,

wherein the selected peptide can be flanked and/or modified at one ofboth termini as described herein. The peptide HBpol 816-824 induces acytotoxic T lymphocyte response which is mediated by at least the MHCclass I molecule HLA-A2.

As mentioned above, additional amino acids can be added to the terminiof an oligopeptide or peptide to provide for ease of linking peptidesone to another, for coupling to a carrier, support or a larger peptide,for reasons discussed herein, or for modifying the physical or chemicalproperties of the peptide or oligopeptide, and the like. Amino acidssuch as tyrosine, cysteine, lysine, glutamic or aspartic acid, and thelike, can be introduced at the C- or N-terminus of the peptide oroligopeptide. In addition, the peptide or oligopeptide sequences candiffer from the natural sequence by being modified by terminal-NH₂acylation, e.g., acetylation, or thioglycolic acid amidation,terminal-carboxy amidation, e.g., ammonia, methylamine, etc. In someinstances these modifications may provide sites for linking to a supportor other molecule.

It will be understood that the HBV peptides of the present invention oranalogs or homologs thereof which have cytotoxic T lymphocytestimulating activity may be modified as necessary to provide certainother desired attributes, e.g., improved pharmacologicalcharacteristics, while increasing or at least retaining substantiallythe biological activity of the unmodified peptide. For instance, thepeptides can be modified by extending, decreasing or substituting aminoacids in the peptide sequence by, e.g., the addition or deletion ofamino acids on either the amino terminal or carboxy terminal end, orboth, of peptides derived from the sequences disclosed herein. Thepeptides may be modified to substantially enhance the CTL inducingactivity, such that the modified peptide analogs have CTL activitygreater than a peptide of the wild-type sequence. For example, it may bedesirable to increase the hydrophobicity of the N-terminal of a peptide,particularly where the second residue of the N-terminal is hydrophobicand is implicated in binding to the HLA restriction molecule. Byincreasing hydrophobicity at the N-terminal, the efficiency of thepresentation to T cells may be increased. Peptides prepared from otherdisease associated antigens, particularly those containing CTL inducingepitopes for which a host may not have significant CTL activity, may bemade CTL-inducing by substituting hydrophobic residues at the N-terminusof the peptide where the second residue is normally hydrophobic.

The peptides employed in the subject invention need not be identical topeptides HBpol61-69 (Seq. ID No. 1),Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val; HBpol 455-463 (SEQ ID NO 2);Gly-Leu-Ser-Arg-T yr-Val-Ala-Arg-Leu; HBpol 773-782 (SEQ ID NO 3),Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val; HBpol803-811 (Seq. ID No. 4),Ser-Leu-Tyr-Ala-Asp-Ser-Pro-Ser-Val; or HBpol816-824 (Seq. ID No. 5),Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu, so long as the subject compoundsare able to provide for cytotoxic T lymphocytic activity against atleast one of the four major subtypes of HBV. Although different strainsof HBV exist, they each share at least one common envelope determinant,which is designated "a". Each strain also has two other envelopedeterminants, one of which is either "d" or "y", and the second iseither "w" or "r". Thus, there are four possible subtypes of the virus:adw, ayw, adr, and ayr. The cloning, sequencing and expression of HBVare described in GB 2034323, EP 13828, U.S. Pat. No. 4,935,235, and thecomplete sequence of the HBV envelope region is also described inGalibert et al., Nature 281:646 (1979), each of the foregoing beingincorporated herein by reference. Amino acid sequences are described inthe GenBank-72 database for 20 different HBV strains, including 7 of theadw subtype, 5 of the ayw subtype, 7 of the adr subtype, and 1 strain ofthe ayr subtype, the GenBank sequences also being incorporated herein byreference.

Therefore, the peptides may be subject to various changes, such asinsertions, deletions, and substitutions, either conservative ornon-conservative, where such changes provide for certain advantages intheir use. By conservative substitutions is meant replacing an aminoacid residue with another which is biologically and/or chemicallysimilar, e.g., one hydrophobic residue for another, or one polar residuefor another. The substitutions include combinations such as Gly, Ala;Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr.Usually, the portion of the sequence which is intended to substantiallymimic an HBV cytotoxic T lymphocyte stimulating epitope will not differby more than about 20% from the sequence of at least one subtype of HBV,except where additional amino acids may be added at either terminus forthe purpose of modifying the physical or chemical properties of thepeptide for, e.g., ease of linking or coupling, and the like. Whereregions of the peptide sequences are found to be polymorphic among HBVsubtypes, it may be desirable to vary one or more particular amino acidsto more effectively mimic differing cytotoxic T-lymphocyte epitopes ofdifferent HBV strains or subtypes.

Within the peptide sequences identified by the present invention,including the representative peptides listed above, there are residues(or those which are substantially functionally equivalent) which allowthe peptide to retain their biological activity, i.e., the ability tostimulate a class I-restricted cytotoxic T-lymphocytic response againstHBV infected cells or cells which express HBV antigen. These residuescan be identified by single amino acid substitutions, deletions, orinsertions. In addition, the contributions made by the side chains ofthe residues can be probed via a systematic scan with a specified aminoacid (e.g., Ala). Peptides which tolerate multiple substitutionsgenerally incorporate such substitutions as small, relatively neutralmolecules, e.g., Ala, Gly, Pro, or similar residues. The number andtypes of residues which can be substituted, added or subtracted willdepend on the spacing necessary between the essential epitopic pointsand certain conformational and functional attributes which are sought(e.g., hydrophobicity vs. hydrophilicity). If desired, increased bindingaffinity of peptide analogues to its MHC molecule for presentation to acytotoxic T-lymphocyte can also be achieved by such alterations.Generally, any spacer substitutions, additions or deletions betweenepitopic and/or conformationally important residues will employ aminoacids or moieties chosen to avoid steric and charge interference whichmight disrupt binding.

Peptides which tolerate multiple substitutions while retaining thedesired biological activity may also be synthesized as D-amino acidcontaining peptides. Such peptide may be synthesized as "inverso" or"retro-inverso" forms, that is, by replacing L-amino acids of a sequencewith D-amino acids, or by reversing the sequence of the amino acids andreplacing the L-amino acids with D-amino acids. As the D-peptides aresubstantially more resistant to peptidases, and therefore are morestable in serum and tissues compared to their L-peptide counterparts,the stability of D-peptides under physiological conditions may more thancompensate for a difference in affinity compared to the correspondingL-peptide. Further, L-amino acid-containing peptides with or withoutsubstitutions can be capped with a D-amino acid to inhibit exopeptidasedestruction of the antigenic peptide.

In addition to the exemplary peptides described herein, the inventionprovides methods for identifying other epitopic regions associated withsaid peptide regions capable of inducing MHC-restricted cytotoxic Tlymphocyte responses against HBV. The methods comprise obtainingperipheral blood lymphocytes (PBL) from infected or uninfectedindividuals and exposing (stimulating) the cells with synthetic peptideor polypeptide fragments derived from a peptide region of HBpol61-69(Seq. ID No. 1), Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val; HBpol 455-463 (SEQID NO 2), Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu; HBpol 773-782 (SEQ ID No3), Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val; HBpol803-811 (Seq. ID No.4), Ser-Leu-Tyr-Ala-Asp-Ser-Pro-Ser-Val; or HBpol816-824 (Seq. ID No.5), Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu. Pools of overlapping syntheticpeptides, each typically about 8 to 20 residues long, preferably 9-12residues, can be used to stimulate the cells. Active peptides can beselected from pools which induce cytotoxic T lymphocyte activity. Theability of the peptides to induce specific cytotoxic activity isdetermined by incubating the stimulated PBL with autologous labeled(e.g., ⁵¹ Cr) target cells (such as HLA matched macrophages, T cells,fibroblasts or B lymphoblastoid cells) infected or transfected with theHBV subgenomic fragments thereof, such that the targeted antigen issynthesized endogenously by the cell (or the cell is pulsed with thepeptide of interest), and measuring specific release of label.

Once a peptide having an epitopic region which stimulates a cytotoxic Tlymphocyte response is identified, the MHC restriction element of theresponse can be determined. This involves incubating the stimulated PBLor short term lines thereof with a panel of (labeled) target cells ofknown HLA types which have been pulsed with the peptide of interest, orappropriate controls. The HLA allele(s) of cells in the panel which arelysed by the CTL are compared to cells not lysed, and the HLArestriction element(s) for the cytotoxic T lymphocyte response to theantigen of interest is identified.

Carbone et al., J. Exp. Med. 167:1767 (1988), have reported thatstimulation with peptides may induce cytotoxic T lymphocytes with lowaffinity for corresponding endogenous protein, such that repetitivepeptide stimulation may yield cytotoxic T lymphocytes that recognizepeptide but not native antigen. As the inability of stimulated cytotoxicT lymphocytes to recognize native HBV proteins would be undesirable inthe development of HBV peptide therapeutics and vaccine compositions,methods to circumvent this potential limitation are used. A sequentialrestimulation of cytotoxic T cells is employed in the present inventionto identify and select T cells with a higher affinity for naturallyprocessed antigen than for a synthetic peptide. Short term cytotoxic Tlymphocyte lines are established by restimulating activated PBL. Cellsstimulated with peptide are restimulated with peptide and recombinant ornative HBV antigen, e.g., HBpol. Cells having activity are alsostimulated with an appropriate T cell mitogen, e.g., phytohemagglutinin(PHA). The restimulated cells are provided with irradiated allogeneicPBLs as an antigen nonspecific source of T cell help, and HBV antigen.To selectively expand the population of cytotoxic T lymphocytes thatrecognize native HBV antigen and to establish long term lines, PBL froma patient are first stimulated with peptide and recombinant or nativeHBV antigen, followed by restimulation with HLA-matched B lymphoblastoidcells that stably express the corresponding HBV antigen polypeptide. Thecell lines are re-confirmed for the ability to recognize endogenouslysynthesized antigen using autologous and allogeneic B-lymphoblastoid orother cells transfected or infected with appropriate antigen.

Having identified different peptides of the invention which contributeto inducing anti-HBV cytotoxic T lymphocyte responses in one or morepatients or HLA types, in some instances it may be desirable to join twoor more peptides in a composition. The peptides in the composition canbe identical or different, and together they should provide equivalentor greater biological activity than the parent peptide(s). For example,using the methods described herein, two or more peptides may definedifferent or overlapping cytotoxic T lymphocyte epitopes from aparticular region, e.g., the HBpol61-69 (Seq. ID No. 1),Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val; HBpol 455-463 (SEQ ID NO 2),Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu; HBpol 773-782 (SEQ ID NO 3),Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val; HBpol803-811 (Seq. ID No. 4),Ser-Leu-Tyr-Ala-Asp-Ser-Pro-Ser-Val; or HBpol816-824 (Seq. ID No. 5),Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu peptides, which peptides can becombined in a "cocktail" to provide enhanced immunogenicity forcytotoxic T lymphocyte responses. Moreover, peptides of one region canbe combined with peptides of other HBV regions, from the same ordifferent HBV protein, particularly when a second or subsequent peptidehas a MHC restriction element different from the first. OtherCTL-inducing HBV peptides are described in co-pending application U.S.Ser. No. 07/935,898 and 08/024,120, which are incorporated herein byreference. This composition of peptides can be used to effectivelybroaden the immunological coverage provided by therapeutic, vaccine ordiagnostic methods and compositions of the invention among a diversepopulation. For example, the different frequencies of HLA alleles amongprevalent ethnic groups (caucasian, asian and african blacks) are shownin Table I below. Therapeutic or vaccine compositions of the inventionmay be formulated to provide potential therapy or immunity to as high apercentage of a population as possible.

                  TABLE I    ______________________________________    HLA ALLELE FREQUENCIES AMONG    PREVALENT ETHNIC GROUPS    HLA Allele   EUC    NAC        AFR  JPN    ______________________________________    A2           45.3   46.6       27.3 43.2    A29          7.4    8.1        12.3 0.4    A31          5.4    6.2        4.4  15.3    A32          8.8    7.1        3    0.1    A33          3.3    3.4        9    13.1    A28*         7.7    9.9        16.6 1.1    ______________________________________     Abbreviations: EUC, European Caucasian; NAC, North American Caucasian;     AFR, African blacks, JPN, Japanese.     *A28 represents the two alleles Aw68 and Aw69

The peptides of the invention can be combined via linkage to formpolymers (multimers), or can be formulated in a composition withoutlinkage, as an admixture. Where the same peptide is linked to itself,thereby forming a homopolymer, a plurality of repeating epitopic unitsare presented. When the peptides differ, e.g., a cocktail representingdifferent HBV subtypes, different epitopes within a subtype, differentHLA restriction specificities, a peptide which contains T helperepitopes, heteropolymers with repeating units are provided. In additionto covalent linkages, noncovalent linkages capable of formingintermolecular and intrastructural bonds are included.

Linkages for homo- or hetero-polymers or for coupling to carriers can beprovided in a variety of ways. For example, cysteine residues can beadded at both the amino- and carboxy-termini, where the peptides arecovalently bonded via controlled oxidation of the cysteine residues.Also useful are a large number of heterobifunctional agents whichgenerate a disulfide link at one functional group end and a peptide linkat the other, including N-succidimidyl-3-(2-pyridyldithio) proprionate(SPDP). This reagent creates a disulfide linkage between itself and acysteine residue in one protein and an amide linkage through the aminoon a lysine or other free amino group in the other. A variety of suchdisulfide/amide forming agents are known. See, for example, Immun. Rev.62:185 (1982), which is incorporated herein by reference. Otherbifunctional coupling agents form a thioether rather than a disulfidelinkage. Many of these thioether forming agents are commerciallyavailable and include reactive esters of 6-maleimidocaproic acid, 2bromoacetic acid, 2-iodoacetic acid, 4-(N-maleimido-methyl) cyclohexane-1-carboxylic acid and the like. The carboxyl groups can be activated bycombining them with succinimide or 1-hydroxy-2-nitro-4-sulfonic acid,sodium salt. A particularly preferred coupling agent is succinimidyl4-(N-maleimidomethyl) cyclohexane- 1-carboxylate (SMCC). It will beunderstood that linkage should not substantially interfere with eitherof the linked groups to function as described, e.g., as an HBV cytotoxicT cell determinant, peptide analogs, or T helper determinant.

In another aspect the peptides of the invention can be combined orcoupled with other peptides which present HBV T-helper cell epitopes,i.e., epitopes which stimulate T cells that cooperate in the inductionof cytotoxic T cells to HBV. The T-helper cells can be either theT-helper 1 or T-helper 2 phenotype, for example. T-helper epitopes fromHBV sequences have been identified at HBc1-20, having the sequence:Met-Asp-Ile-Asp-Pro-Tyr-Lys-Glu-Phe-Gly-Ala-Thr-Val-Glu-Leu-Leu-Ser-Phe-Leu-Pro(Seq. ID No, 6). Other T-helper epitopes are provided by peptides fromthe region HBc50-69, having the sequencePro-His-His-Tyr-Ala-Leu-Arg-Gln-Ala-Ile-Leu-Cys-Trp-Gly-Glu-Leu-Met-Tyr-Leu-Ala(Seq. ID No. 7), and from the region of HBc100-139, including HBc100-119having the sequenceLeu-Leu-Trp-Phe-His-Ile-Ser-Cys-Leu-Thr-Phe-Gly-Arg-Glu-Thr-Val-Ile-Glu-Tyr-Leu(Seq. ID No. 8) (where Ile₁₁₆ is Leu in the HBV adw subtype), HBc117-131having the sequenceGlu-Tyr-Leu-Val-Ser-Phe-Gly-Val-Trp-Ile-Arg-Thr-Pro-Pro-Ala (Seq. ID No.9), and peptide HBc120-139 having the sequenceVal-Ser-Phe-Gly-Val-Trp-Ile-Arg-Thr-Pro-Pro-Ala-Tyr-Arg-Pro-Pro-Asn-Ala-Pro-Ile(Seq. ID No. 10). See, Ferrari et al., J. Clin. Invest. 88:214-222(1991), and U.S. Pat. 4,882,145, each incorporated herein by reference.

The peptides of the invention can be prepared in a wide variety of ways.Because of their relatively short size, the peptides can be synthesizedin solution or on a solid support in accordance with conventionaltechniques. Various automatic synthesizers are commercially availableand can be used in accordance with known protocols. See, for example,Stewart and Young, Solid Phase Peptide Synthesis, 2d. ed., PierceChemical Co. (1984); Tam et al., J. Am. Chem. Soc. 105:6442 (1983);Merrifield, Science 232:341-347 (1986); and Barany and Merrifield, ThePeptides, Gross and Meienhofer, eds., Academic Press, New York, pp.1-284 (1979), each of which is incorporated herein by reference.

Alternatively, recombinant DNA technology may be employed wherein anucleotide sequence which encodes a peptide of interest is inserted intoan expression vector, transformed or transfected into an appropriatehost cell and cultivated under conditions suitable for expression. Theseprocedures are generally known in the art, as described generally inSambrook et al., Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Press, Cold Spring Harbor, New York (1982), and Ausubel et al.,(ed.) Current Protocols in Molecular Biology, John Wiley and Sons, Inc.,New York (1987), and U.S. Pat. Nos. 4,237,224, 4,273,875, 4,431,739,4,363,877 and 4,428,941, for example, whose disclosures are eachincorporated herein by reference. Thus, fusion proteins which compriseone or more peptide sequences of the invention can be used to presentthe HBV cytotoxic T cell determinants. For example, a recombinantpolymerase protein of the invention is prepared in which the HBpol aminoacid sequence is altered so as to more effectively present epitopes ofpeptide regions described herein to stimulate a cytotoxic T lymphocyteresponse. By this means a polypeptide is used which incorporates severalT cell epitopes.

As the coding sequence for peptides of the length contemplated hereincan be synthesized by chemical techniques, for example, thephosphotriester method of Matteucci et al., J. Am. Chem. Soc, 103:3185(1981), modification can be made simply by substituting the appropriatebase(s) for those encoding the native peptide sequence. The codingsequence can then be provided with appropriate linkers and ligated intoexpression vectors commonly available in the art, and the vectors usedto transform suitable hosts to produce the desired fusion protein. Anumber of such vectors and suitable host systems are now available. Forexpression of the fusion proteins, the coding sequence will be providedwith operably linked start and stop codons, promoter and terminatorregions and usually a replication system to provide an expression vectorfor expression in the desired cellular host. For example, promotersequences compatible with bacterial hosts are provided in plasmidscontaining convenient restriction sites for insertion of the desiredcoding sequence. The resulting expression vectors are transformed intosuitable bacterial hosts. Yeast or mammalian cell hosts may also beused, employing suitable vectors and control sequences.

The peptides of the present invention and pharmaceutical and vaccinecompositions thereof are useful for administration to mammals,particularly humans, to treat and/or prevent HBV infection. As thepeptides are used to stimulate cytotoxic T-lymphocyte responses to HBVinfected cells, the compositions can be used to treat or prevent acuteand/or chronic HBV infection.

For pharmaceutical compositions, the peptides of the invention asdescribed above will be administered to an individual already infectedwith HBV. Those in the incubation phase or the acute phase of infectioncan be treated with the immunogenic peptides separately or inconjunction with other treatments, as appropriate. In therapeuticapplications, compositions are administered to a patient in an amountsufficient to elicit an effective cytotoxic T lymphocyte response to HBVand to cure or at least partially arrest its symptoms and/orcomplications. An amount adequate to accomplish this is defined as"therapeutically effective dose." Amounts effective for this use willdepend on, e.g., the peptide composition, the manner of administration,the stage and severity of the disease being treated, the weight andgeneral state of health of the patient, and the judgment of theprescribing physician, but generally range from about 1 μg to about2,000 mg of peptide for a 70 kg patient, with dosages of from about 10μg to about 100 mg of peptide being more commonly used, followed bybooster dosages from about 1 μg to about 1 mg of peptide over weeks tomonths, depending on a patient's CTL response, as determined bymeasuring HBV-specific CTL activity in PBLs obtained from the patient.It must be kept in mind that the peptides and compositions of thepresent invention may generally be employed in serious disease states,that is, life-threatening or potentially life threatening situations. Insuch cases, in view of the minimization of extraneous substances and therelative nontoxic nature of the peptides, it is possible and may be feltdesirable by the treating physician to administer substantial excessesof these peptide compositions.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical formulations should providea quantity of cytotoxic T-lymphocyte stimulatory peptides of theinvention sufficient to effectively treat the patient.

For therapeutic use, administration should begin at the first sign ofHBV infection or shortly after diagnosis in cases of acute infection,and continue until at least symptoms are substantially abated and for aperiod thereafter. In well established and chronic cases, loading dosesfollowed by maintenance or booster doses may be required. Theelicitation of an effective cytotoxic T lymphocyte response to HBVduring treatment of acute hepatitis will minimize the possibility ofsubsequent development of chronic hepatitis, HBV carrier stage, andensuing hepatocellular carcinoma.

Treatment of an infected individual with the compositions of theinvention may hasten resolution of the infection in acutely infectedindividuals, about 90% of whom are capable of resolving the infectionnaturally. For those individuals susceptible (or predisposed) todeveloping chronic infection the compositions are particularly useful inmethods for preventing the evolution from acute to chronic infection.Where the susceptible individuals are identified prior to or duringinfection, for instance, as described herein, the composition can betargeted to them, minimizing need for administration to a largerpopulation.

The peptide compositions can also be used for the treatment of chronichepatitis and to stimulate the immune system of carriers tosubstantially reduce or even eliminate virus-infected cells. Those withchronic hepatitis can be identified as testing positive for virus fromabout 3-6 months after infection. As individuals may develop chronic HBVinfection because of an inadequate (or absent) cytotoxic T lymphocyteresponse during the acute phase of their infection, it is important toprovide an amount of immuno-potentiating peptide in a formulation andmode of administration sufficient to effectively stimulate a cytotoxic Tcell response. Thus, for treatment of chronic hepatitis, arepresentative dose is in the range of about 1 μg to 1,000 mg,preferably about 5 μg to 100 mg for a 70 kg patient per dose.Administration should continue until at least clinical symptoms orlaboratory indicators indicate that the HBV infection has beeneliminated or substantially abated and for a period thereafter.Immunizing doses followed by maintenance or booster doses at establishedintervals, e.g., from one to four weeks, may be required, possibly for aprolonged period of time, as necessary to resolve the infection. For thetreatment of chronic and carrier HBV infection it may also be desirableto combine the CTL peptides with other peptides or proteins that induceimmune response to other HBV antigens.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral or local administration. Preferably, thepharmaceutical compositions are administered parenterally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration whichcomprise a solution of the cytotoxic T-lymphocyte stimulatory peptidesdissolved or suspended in an acceptable carrier, preferably an aqueouscarrier. A variety of aqueous carriers may be used, e.g., water,buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.These compositions may be sterilized by conventional, well knownsterilization techniques, or may be sterile filtered. The resultingaqueous solutions may be packaged for use as is, or lyophilized, thelyophilized preparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc.

In some embodiments it may be desirable to include in the pharmaceuticalcomposition at least one component which primes CTL. Lipids have beenidentified which are capable of priming CTL in vivo against viralantigens, e.g., tripalmitoyl-S-glycerylcysteinyl-seryl-serine (P₃ CSS),which can effectively prime virus specific cytotoxic T lymphocytes whencovalently attached to an appropriate peptide. See, Deres et al., Nature342:561-564 (1989), incorporated herein by reference. Peptides of theinvention can be coupled to P₃ CSS, for example, and the lipopeptideadministered to an individual to specifically prime a cytotoxic Tlymphocyte response to HBV. Further, as the induction of neutralizingantibodies can also be primed with P₃ CSS conjugated to a peptide whichdisplays an appropriate epitope, e.g., HBsAg epitopes, the twocompositions can be combined to more effectively elicit both humoral andcell-mediated responses to HBV infection.

The concentration of cytotoxic T-lymphocyte stimulatory peptides of theinvention in the pharmaceutical formulations can vary widely, i.e., fromless than about 1%, usually at or at least about 10% to as much as 20 to50% or more by weight, and will be selected primarily by fluid volumes,viscosities, etc., in accordance with the particular mode ofadministration selected.

Thus, a typical pharmaceutical composition for intravenous infusioncould be made up to contain 250 ml of sterile Ringer's solution, and 100mg of peptide. Actual methods for preparing parenterally administrablecompounds will be known or apparent to those skilled in the art and aredescribed in more detail in for example, Remington's PharmaceuticalScience, 17th ed., Mack Publishing Company, Easton, Pa. (1985), which isincorporated herein by reference.

The peptides of the invention may also be administered via liposomes,which serve to target the peptides to a particular tissue, such aslymphoid tissue or HBV-infected hepatic cells. Liposomes can also beused to increase the half-life of the peptide composition. Liposomesuseful in the present invention include emulsions, foams, micelles,insoluble monolayers, liquid crystals, phospholipid dispersions,lamellar layers and the like. In these preparations the peptide to bedelivered is incorporated as part of a liposome, alone or in conjunctionwith a molecule which binds to, e.g., a receptor, prevalent amonglymphoid cells, such as monoclonal antibodies which bind to the CD45antigen, or with other therapeutic or immunogenic compositions. Thus,liposomes filled with a desired peptide of the invention can be directedto the site of lymphoid or hepatic cells, where the liposomes thendeliver the selected therapeutic/immunogenic peptide compositions.Liposomes for use in the invention are formed from standardvesicle-forming lipids, which generally include neutral and negativelycharged phospholipids and a sterol, such as cholesterol. The selectionof lipids is generally guided by consideration of, e.g., liposome sizeand stability of the liposomes in the blood stream. A variety of methodsare available for preparing liposomes, as described in, e.g., Szoka etal., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871,4,501,728, 4,837,028, and 5,019,369, incorporated herein by reference.For targeting to the immune cells, a ligand to be incorporated into theliposome can include, e.g., antibodies or fragments thereof specific forcell surface determinants of the desired immune system cells. A liposomesuspension containing a peptide may be administered intravenously,locally, topically, etc. in a dose which varies according to, the modeof administration, the peptide being delivered, the stage of diseasebeing treated, etc.

For solid compositions, conventional nontoxic solid carriers may be usedwhich include, for example, pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharin, talcum, cellulose,glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

For aerosol administration, the cytotoxic T-lymphocyte stimulatorypeptides are preferably supplied in finely divided form along with asurfactant and propellant. Typical percentages of peptides are 0.01%-20%by weight, preferably 1%-10%. The surfactant must, of course, benontoxic, and preferably soluble in the propellant. Representative ofsuch agents are the esters or partial esters of fatty acids containingfrom 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic,stearic, linoleic, linolenic, olesteric and oleic acids with analiphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, suchas mixed or natural glycerides may be employed. The surfactant mayconstitute 0.1%-20% by weight of the composition, preferably 0.25-5%.The balance of the composition is ordinarily propellant. A carrier canalso be included as desired, e.g., lecithin for intranasal delivery.

In another aspect the present invention is directed to vaccines whichcontain as an active ingredient an immunogenically effective amount of acytotoxic T-lymphocyte stimulating peptide as described herein. Thepeptide(s) may be introduced into a host, including humans, linked toits own carrier or as a homopolymer or heteropolymer of active peptideunits. Such a polymer has the advantage of increased immunologicalreaction and, where different peptides are used to make up the polymer,the additional ability to induce antibodies and/or cytotoxic T cellsthat react with different antigenic determinants of HBV. Useful carriersare well known in the art, and include, e.g., keyhole limpet hemocyanin,thyroglobulin, albumins such as human serum albumin, tetanus toxoid,polyamino acids such as poly(D-lysine:D-glutamic acid), and the like.The vaccines can also contain a physiologically tolerable (acceptable)diluent such as water, phosphate buffered saline, or saline, and furthertypically include an adjuvant. Adjuvants such as incomplete Freund'sadjuvant, aluminum phosphate, aluminum hydroxide, or alum are materialswell known in the art. And, as mentioned above, cytotoxic T lymphocyteresponses can be primed by conjugating peptides of the invention tolipids, such as P₃ CSS. Upon immunization with a peptide composition asdescribed herein, via injection, aerosol, oral, transdermal or otherroute, the immune system of the host responds to the vaccine byproducing large amounts of cytotoxic T-lymphocytes specific for HBVantigen, and the host becomes at least partially immune to HBVinfection, or resistant to developing chronic HBV infection.

Vaccine compositions containing the peptides of the invention areadministered to a patient susceptible to or otherwise at risk of HBVinfection to enhance the patient's own immune response capabilities.Such an amount is defined to be a "immunogenically effective dose." Inthis use, the precise amounts again depend on the patient's state ofhealth and weight, the mode of administration, the nature of theformulation, etc., but generally range from about 1.0 μg to about 500 mgper 70 kilogram patient, more commonly from about 50 μg to about 200 mgper 70 kg of body weight. The peptides are administered to individualsof an appropriate HLA type, e.g., for vaccine compositions of peptidesfrom the region of HBpol61-69 (Seq. ID No. 1),Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val; HBpol 455-463 (SEQ ID NO 2),Gly-Leu-Ser-Arg-Tyr-Val-Ala-Arg-Leu; HBpol 773-782 (SEQ ID NO 3),Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val; HBpol803-811 (Seq. ID No. 4),Ser-Leu-Tyr-Ala-Asp-Ser-Pro-Ser-Val; or HBpol816-824 (Seq. ID No. 5),Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu, these will be administered to atleast HLA-A2 individuals.

In some instances it may be desirable to combine the peptide vaccines ofthe invention with vaccines which induce neutralizing antibody responsesto HBV, particularly to HBV envelope and/or core antigens, such asrecombinant HBV env- and/or nucleocapsid-encoded antigens or vaccinesprepared from purified plasma preparations obtained from HBV-infectedindividuals. A variety of HBV vaccine preparations have been described,and are based primarily on HBsAg and polypeptide fragments thereof. Forexamples of vaccines which can be formulated with the peptides of thepresent invention, see generally, EP 154,902 and EP 291,586, and U.S.Pat. Nos. 4,565,697, 4,624,918, 4,599,230, 4,599,231, 4,803,164,4,882,145, 4,977,092, 5,017,558 and 5,019,386, each being incorporatedherein by reference. The vaccines can be combined and administeredconcurrently, or as separate preparations.

For therapeutic or immunization purposes, the peptides of the inventioncan also be expressed by attenuated viral hosts, such as vaccinia. Thisapproach involves the use of vaccinia virus as a vector to expressnucleotide sequences that encode the HBV peptides of the invention. Uponintroduction into an acutely or chronically HBV-infected host or into anon-infected host, the recombinant vaccinia virus expresses the HBVpeptide and thereby elicits a host cytotoxic T lymphocyte response toHBV. Vaccinia vectors and methods useful in immunization protocols aredescribed in, e.g., U.S. Pat. No. 4,722,848, incorporated herein byreference. Another vector is BCG (bacille Calmette Guerin). BCG vectorsare described in Stover et al. (Nature 351:456-460 (1991)) which isincorporated herein by reference. A wide variety of other vectors usefulfor therapeutic administration or immunization of the peptides of theinvention, e.g., Salmonella typhi vectors and the like, will be apparentto those skilled in the art from the description herein.

The compositions and methods of the claimed invention may be employedfor ex vivo therapy. By ex vivo therapy is meant that therapeutic orimmunogenic manipulations are performed outside the body. For example,lymphocytes or other target cells may be removed from a patient andtreated with high doses of the subject peptides, providing a stimulatoryconcentration of peptide in the cell medium far in excess of levelswhich could be accomplished or tolerated by the patient. Followingtreatment to stimulate the CTLs, the cells are returned to the host totreat the HBV infection. The host's cells may also be exposed to vectorswhich carry genes encoding the peptides, as described above. Oncetransfected with the vectors, the cells may be propagated in vitro orreturned to the patient. The cells which are propagated in vitro may bereturned to the patient after reaching a predetermined cell density.

In one method, ex vivo CTL responses to a HBV are induced by incubatingin tissue culture a patient's CTL precursor cells (CTLp) together with asource of antigen-presenting cells (APC) and the appropriate immunogenicpeptide. After an appropriate incubation time (typically 1-4 weeks), inwhich the CTLp are activated and mature and expand into effector CTL,the cells are infused back into the patient, where they will destroytheir specific target cell (an HBV infected cell). To optimize the invitro conditions for the generation of specific cytotoxic T cells, theculture of stimulator cells is typically maintained in an appropriateserum-free medium. Peripheral blood lymphocytes are convenientlyisolated following simple venipuncture or leukapheresis of normal donorsor patients and used as the responder cell sources of CTLp. In oneembodiment, the appropriate APC are incubated with about 10-100 μM ofpeptide in serum-free media for 4 hours under appropriate cultureconditions. The peptide-loaded APC are then incubated with the respondercell populations in vitro for 5 to 10 days under optimized cultureconditions.

Positive CTL activation can be determined by assaying the cultures forthe presence of CTLs that kill radiolabeled target cells, both specificpeptide-pulsed targets as well as target cells expressing endogenouslyprocessed form of the HBV polymerase antigen from which the peptidesequence was derived. Specificity and MHC restriction of the CTL of apatient can be determined by a number of methods known in the art. Forinstance, CTL restriction can be determined by testing against differentpeptide target cells expressing appropriate or inappropriate human MHCclass I. The peptides that test positive in the MHC binding assays andgive rise to specific CTL responses are identified as immunogenicpeptides.

The induction of CTL in vitro requires the specific recognition ofpeptides that are bound to allele specific MHC class I molecules on APC.Peptide loading of empty major histocompatibility complex molecules oncells allows the induction of primary CTL responses. Since mutant celllines do not exist for every human MHC allele, it may be advantageous touse a technique to remove endogenous MHC-associated peptides from thesurface of APC, followed by loading the resulting empty MHC moleculeswith the immunogenic peptides of interest. The use of non-transformed,non-infected cells, and preferably, autologous cells of patients as APCis desirable for the design of CTL induction protocols directed towardsdevelopment of ex vivo CTL therapies. Typically, prior to incubation ofthe APCs with the CTLp to be activated, an amount of antigenic peptideis added to the APC or stimulator cell culture, of sufficient quantityto become loaded onto the human Class I molecules to be expressed on thesurface of the APCs. Resting or precursor CTLs are then incubated inculture with the appropriate APCs for a time period sufficient toactivate the CTLs. Preferably, the CTLs are activated in anantigen-specific manner. The ratio of resting or precursor CTLs to APCsmay vary from individual to individual and may further depend uponvariables such as the amenability of an individual's lymphocytes toculturing conditions and the nature and severity of the diseasecondition or other condition for which the described treatment modalityis used. Preferably, however, the CTL:APC ratio is in the range of about30:1 to 300:1. The CTL/APC may be maintained for as long a time as isnecessary to stimulate a therapeutically useable or effective number ofCTL.

Activated CTL may be effectively separated from the APC using one of avariety of known methods. For example, monoclonal antibodies specificfor the APCs, for the peptides loaded onto the stimulator cells, or forthe CTL (or a segment thereof) may be utilized to bind their appropriatecomplementary ligand. Antibody-tagged molecules may then be extractedfrom the admixture via appropriate means, e.g., via well-knownimmunoprecipitation or immunoassay methods.

Effective, cytotoxic amounts of the activated CTLs can vary between invitro and in vivo uses, as well as with the amount and type of cellsthat are the ultimate target of these killer cells. The amount will alsovary depending on the condition of the patient and should be determinedvia consideration of all appropriate factors by the practitioner.Preferably, however, about 1×10⁶ to about 1×10¹², more preferably about1×10⁸ to about 1×10¹¹, and even more preferably, about 1×10⁹ to about1×10¹⁰ activated CD8+ cells are utilized for adult humans, compared toabout 5×10⁶ -5×10⁷ cells used in mice.

Methods of reintroducing cellular components are known in the art andinclude procedures such as those exemplified in U.S. Pat. No. 4,844,893to Honsik, et al. and U.S. Pat. No. 4,690,915 to Rosenberg, which areincorporated herein by reference. For example, administration ofactivated CTLs via intravenous infusion is typically appropriate.

The peptides may also find use as diagnostic reagents. For example, apeptide of the invention may be used to determine the susceptibility ofa particular individual to a treatment regimen which employs the peptideor related peptides, and thus may be helpful in modifying an existingtreatment protocol or in determining a prognosis for an affectedindividual. In addition, the peptides may also be used to predict whichindividuals will be at substantial risk for developing chronic HBVinfection.

The following examples are offered by way of illustration, not by way oflimitation.

EXAMPLE I HLA-Restricted CTL Response TO HBV Polymerase Epitopes

This Example describes the identification of an HLA-A2 restricted CTLresponse to two HBV polymerase peptides in a patient with acute viralhepatitis. The epitopes are present in amino acid sequences HBpol₆₁₋₆₉Seq. ID No. 1! Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val (GLYSSTVPV) (alsodesignated peptide 927.32) and HBpol₈₀₃₋₁₈₁₁ Seq. ID No. 4!Ser-Leu-Tyr-Ala-Asp-Ser-Pro-Ser-Val (SLYADSPSV) (also designated peptide927.27).

The CTL induced by the HBpol peptides were identified in PBMCs from apatient with acute hepatitis according to the procedure set forth inExample VI of pending application U.S. Ser. No. 07/935,898, except thatthe PMBCs were stimulated with individual peptides rather than peptidemixtures. The resulting CTL lines and/or clones were then tested for theability to kill HLA-A2 matched target cells that were either pulsed withthe peptide or that expressed the corresponding endogenous polymeraseantigen (Vpol or EBO-pol).

Construction of the vaccinia based Vpol and Epstein-Barr virus basedEBO-pol constructs was as described in Example II of U.S. Ser. No.07/935,898.

As shown in FIG. 1, both peptides HBpol₈₀₃₋₈₁₁ and HBpol₆₁₋₆₉ stimulatedCTL responses in a patient (HLA-A2⁺) using target cells pulsed withpeptide, whereas other peptides 927.24 (WILRGTSFR) and 927.30(DLNLGNLNV) and media controls did not stimulate the specific CTLresponse. The ability of the HBpol₈₀₃₋₈₁₁ specific clones to recognizeendogenously synthesized polymerase antigen (Vpol and EBO-pol) is shownin FIG. 2. Two clones, designated Be.27-1A1 and Be.27-1A5, wereidentified that recognized the HBpol₈₀₃₋₈₁₁ peptide. As shown in FIG. 3,CTL responses to HBpol₆₁₋₆₉ and HBpol₈₀₃₋₈₁₁ were shown with targetcells pulsed with homologous peptide, but only the HBpol₈₀₃₋₈₁₁ cloneshowed a response to endogenously synthesized Vpol antigen.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

From the foregoing it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES: 11    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 9 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    -      Gly Leu Tyr Ser Ser Thr Val Pro - # Val    #  5 1    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 9 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    -      Gly Leu Ser Arg Tyr Val Ala Arg - # Leu    #  5 1    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 10 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    -      Ile Leu Arg Gly Thr Ser Phe Val - # Tyr Val    #   10    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 9 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    -      Ser Leu Tyr Ala Asp Ser Pro Ser - # Val    #  5 1    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 9 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    -      Phe Leu Leu Ser Leu Gly Ile His - # Leu    #  5 1    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 20 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    -      Met Asp Ile Asp Pro Tyr Lys Glu - # Phe Gly Ala Thr Val Glu Leu    Leu    #   15    -      Ser Phe Leu Pro                     20    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 20 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    -      Pro His His Tyr Ala Leu Arg Gln - # Ala Ile Leu Cys Trp Gly Glu    Leu    #   15    -      Met Tyr Leu Ala                     20    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 20 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    -      Leu Leu Trp Phe His Ile Ser Cys - # Leu Thr Phe Gly Arg Glu Thr    Val    #   15    -      Ile Glu Tyr Leu                     20    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 15 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    -      Glu Tyr Leu Val Ser Phe Gly Val - # Trp Ile Arg Thr Pro Pro Ala    #   15    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 20 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    -      Val Ser Phe Gly Val Trp Ile Arg - # Thr Pro Pro Ala Tyr Arg Pro    Pro    #   15    -      Asn Ala Pro Ile                     20    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 845 amino              (B) TYPE: amino acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    -      Met Pro Leu Ser Tyr Gln His Phe - # Arg Lys Leu Leu Leu Leu Asp    Asp    #   15    -      Gly Thr Glu Ala Gly Pro Leu Glu - # Glu Glu Leu Pro Arg Leu Ala    Asp    #                 30    -      Glu Gly Leu Asn Arg Arg Val Ala - # Glu Asp Leu Asn Leu Gly Asn    Leu    #             45    -      Asn Val Ser Ile Pro Trp Thr His - # Lys Val Gly Asn Phe Thr Gly    Leu    #         60    -      Tyr Ser Ser Thr Val Pro Val Phe - # Asn Pro Glu Trp Gln Thr Pro    Ser    #     80    -      Phe Pro Asn Ile His Leu Gln Glu - # Asp Ile Ile Asn Arg Cys Gln    Gln    #   95    -      Phe Val Gly Pro Leu Thr Val Asn - # Glu Lys Arg Arg Leu Lys Leu    Ile    #                110    -      Met Pro Ala Arg Phe Tyr Pro Asn - # Val Thr Lys Tyr Leu Pro Leu    Asp    #            125    -      Lys Gly Ile Lys Pro Tyr Tyr Pro - # Glu His Leu Val Asn His Tyr    Phe    #        140    -      Gln Thr Arg His Tyr Leu His Thr - # Leu Trp Lys Ala Gly Ile Leu    Tyr    #    160    -      Lys Arg Glu Thr Thr Arg Ser Ala - # Ser Phe Cys Gly Ser Pro Tyr    Ser    #   175    -      Trp Glu Gln Glu Leu Gln His Gly - # Arg Leu Val Phe Gln Thr Ser    Thr    #                190    -      Arg His Gly Asp Glu Ser Phe Cys - # Ser Gln Ser Ser Gly Ile Leu    Ser    #            205    -      Arg Ser Pro Val Gly Pro Cys Ile - # Arg Ser Gln Leu Arg Gln Ser    Arg    #        220    -      Leu Gly Leu Gln Pro Gln Gln Gly - # His Leu Ala Arg Arg Gln Gln    Gly    #    240    -      Arg Ser Gly Ser Ile Arg Ala Arg - # Val His Pro Thr Thr Arg Arg    Ser    #   255    -      Phe Gly Val Glu Pro Ser Gly Ser - # Gly His Ile Asp Asn Ser Ala    Ser    #                270    -      Ser Ser Ser Ser Cys Leu His Gln - # Ser Ala Val Arg Lys Ala Ala    Tyr    #            285    -      Ser His Leu Ser Thr Ser Lys Arg - # Gln Ser Ser Ser Gly His Ala    Val    #        300    -      Glu Leu His Asn Ile Pro Pro Asn - # Ser Ala Arg Ser Gln Ser Glu    Gly    #    320    -      Pro Val Phe Ser Cys Trp Trp Leu - # Gln Phe Arg Asn Ser Lys Pro    Cys    #   335    -      Ser Asp Tyr Cys Leu Thr His Ile - # Val Asn Leu Leu Glu Asp Trp    Gly    #                350    -      Pro Cys Thr Glu His Gly Glu His - # Asn Ile Arg Ile Pro Arg Thr    Pro    #            365    -      Ala Arg Val Thr Gly Gly Val Phe - # Leu Val Asp Lys Asn Pro His    Asn    #        380    -      Thr Thr Glu Ser Arg Leu Val Val - # Asp Phe Ser Gln Phe Ser Arg    Gly    #    400    -      Ser Thr Arg Val Ser Trp Pro Lys - # Phe Ala Val Pro Asn Leu Gln    Ser    #   415    -      Leu Thr Asn Leu Leu Ser Ser Asn - # Leu Ser Trp Leu Ser Leu Asp    Val    #                430    -      Ser Ala Ala Phe Tyr His Ile Pro - # Leu His Pro Ala Ala Met Pro    His    #            445    -      Leu Leu Val Gly Ser Ser Gly Leu - # Ser Arg Tyr Val Ala Arg Leu    Ser    #        460    -      Ser Asn Ser Arg Ile Ile Asn Tyr - # Gln His Gly Thr Met Gln Asn    Leu    #    480    -      His Asp Ser Cys Ser Arg Asn Leu - # Tyr Val Ser Leu Leu Leu Leu    Tyr    #   495    -      Lys Thr Phe Gly Arg Lys Leu His - # Leu Tyr Ser His Pro Ile Ile    Leu    #                510    -      Gly Phe Arg Lys Ile Pro Met Gly - # Val Gly Leu Ser Pro Phe Leu    Leu    #            525    -      Ala Gln Phe Thr Ser Ala Ile Cys - # Ser Val Val Arg Arg Ala Phe    Pro    #        540    -      His Cys Leu Ala Phe Ser Tyr Met - # Asp Asp Val Val Leu Gly Ala    Lys    #    560    -      Ser Val Gln His Leu Glu Ser Leu - # Phe Thr Ala Val Thr Asn Phe    Leu    #   575    -      Leu Ser Leu Gly Ile His Leu Asn - # Pro Asn Lys Thr Lys Arg Trp    Gly    #                590    -      Tyr Ser Leu Asn Phe Met Gly Tyr - # Val Ile Gly Ser Trp Gly Thr    Ile    #            605    -      Pro Gln Glu His Ile Val Gln Lys - # Ile Lys Gln Cys Phe Arg Lys    Leu    #        620    -      Pro Val Asn Arg Pro Ile Asp Trp - # Lys Val Cys Gln Arg Ile Val    Gly    #    640    -      Leu Leu Gly Phe Ala Ala Pro Phe - # Thr Gln Cys Gly Tyr Pro Ala    Leu    #   655    -      Met Pro Leu Tyr Ala Cys Ile Gln - # Ser Lys Gln Ala Phe Thr Phe    Ser    #                670    -      Pro Thr Tyr Lys Ala Phe Leu Cys - # Lys Gln Tyr Leu Asn Leu Tyr    Pro    #            685    -      Val Ala Arg Gln Arg Pro Gly Leu - # Cys Gln Val Phe Ala Asp Ala    Thr    #        700    -      Pro Thr Gly Trp Gly Leu Ala Ile - # Gly His Gln Arg Met Arg Gly    Thr    #    720    -      Phe Val Ala Pro Leu Pro Ile His - # Thr Ala Glu Leu Leu Ala Ala    Cys    #   735    -      Phe Ala Arg Ser Arg Ser Gly Ala - # Lys Leu Ile Gly Thr Asp Asn    Ser    #                750    -      Val Val Leu Ser Arg Lys Tyr Thr - # Ser Phe Pro Trp Leu Leu Gly    Cys    #            765    -      Ala Ala Asn Trp Ile Leu Arg Gly - # Thr Ser Phe Val Tyr Val Pro    Ser    #        780    -      Ala Leu Asn Pro Ala Asp Asp Pro - # Ser Arg Gly Arg Leu Gly Leu    Tyr    #    800    -      Arg Pro Leu Leu Arg Leu Pro Phe - # Arg Pro Thr Thr Gly Arg Thr    Ser    #   815    -      Leu Tyr Ala Val Ser Pro Ser Val - # Pro Ser His Leu Pro Asp Arg    Val    #                830    -      His Phe Ala Ser Pro Leu His Val - # Ala Trp Arg Pro Pro    #            845    __________________________________________________________________________

What is claimed is:
 1. A peptide comprising from eight to thirteen aminoacids and having at least seven contiguous amino acids of acorresponding portion of HBpol having the sequence:

          (HBpol.sub.61-69) (SEQ ID No. 1)    Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val

wherein said peptide binds with an appropriate HLA molecule to form acomplex recognized by cytotoxic T cells which T cells recognize a nativeHBV antigen.
 2. The peptide of claim 1, which comprises from nine toeleven amino acids.
 3. The peptide of claim 1, which comprises:

          (HBpol.sub.61-69) (SEQ ID No. 1)    Gly-Leu-Tyr-Ser-Ser-Thr-Val-Pro-Val.


4. An immunogenic composition comprising the peptide of claim 1 and apharmaceutically acceptable lipid-containing carrier.
 5. The immunogeniccomposition of claim 4, wherein the lipid-containing carrier enhances ahuman T-lymphocyte response.
 6. The immunogenic composition of claim 4,wherein the lipid-containing carrier comprises a lipoprotein.
 7. Theimmunogenic composition of claim 4, wherein the lipid-containing carriercomprises a liposome.
 8. An immunogenic composition comprising thepeptide of claim 1 and a second immunogenic peptide.
 9. The immunogeniccomposition of claim 8, wherein the second immunogenic peptide elicitsan immune response specific for hepatitis B virus.
 10. The immunogeniccomposition of claim 8, wherein the second immunogenic peptide elicits aT-helper cell mediated response.
 11. An immunogenic compositioncomprising the peptide of claim 1 and a second immunogenic peptideconjugated to form a heteropolymer.
 12. An immunogenic compositioncomprising the peptide of claim 1, conjugated to a lipid carrier. 13.The peptide of claim 1, which is expressed by a DNA construct comprisinga transcriptional promotor, a DNA sequence encoding said peptide, and atranscription terminator, each operably linked for expression of saidpeptide.
 14. A peptide comprising from eight to thirteen amino acids andhaving at least seven contiguous amino acids of a corresponding portionof HBpol having the sequence:

            (HBpol.sub.773-782) (SEQ ID No. 3)    Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val

wherein said peptide binds with an appropriate HLA molecule to form acomplex recognized by cytotoxic T cells which T cells recognize a nativeHBV antigen.
 15. The peptide of claim 14, which comprises from nine toeleven amino acids.
 16. The peptide of claim 14, which comprises:

                  (HBpol.sub.773-782) (SEQ ID No. 3)    Ile-Leu-Arg-Gly-Thr-Ser-Phe-Val-Tyr-Val.


17. An immunogenic composition comprising the peptide of claim 14 and asecond immunogenic peptide.
 18. The immunogenic composition of claim 17,wherein the second immunogenic peptide elicits an immune responsespecific for hepatitis B virus.
 19. The immunogenic composition of claim17, wherein the second immunogenic peptide elicits a T-helper cellmediated response.
 20. An immunogenic composition comprising the peptideof claim 14 and a second immunogenic peptide conjugated to form aheteropolymer.
 21. An immunogenic composition comprising the peptide ofclaim 14 and a pharmaceutically acceptable lipid-containing carrier. 22.The immunogenic composition of claim 21, wherein the lipid-containingcarrier enhances a human T-lymphocyte response.
 23. The immunogeniccomposition of claim 21, wherein the lipid-containing carrier comprisesa lipoprotein.
 24. The immunogenic composition of claim 21, wherein thelipid-containing carrier comprises a liposome.
 25. The peptide of claim14, which is expressed by a DNA construct comprising a transcriptionalpromotor, a DNA sequence encoding said peptide, and a transcriptionterminator, each operably linked for expression of said peptide.
 26. Apeptide comprising from eight to thirteen amino acids and having atleast seven contiguous amino acids of a corresponding portion of HBpolhaving the sequence:

                (HBpol.sub.803-811)    Ser-Leu-Tyr-Ala-Asp-Ser-Pro-Ser-Val (SEQ ID No. 4)

wherein said peptide binds with an appropriate HLA molecule to form acomplex recognized by cytotoxic T cells which T cells recognize a nativeHBV antigen.
 27. The peptide of claim 26, which comprises from nine toeleven amino acids.
 28. The peptide of claim 26, which comprises:

          (HBpol.sub.803-811) (SEQ ID No. 4)    Ser-Leu-Tyr-Ala-Asp-Ser-Pro-Ser-Val.


29. An immunogenic composition comprising the peptide of claim 26 and asecond immunogenic peptide.
 30. The immunogenic composition of claim 29,wherein the second immunogenic peptide elicits an immune responsespecific for hepatitis B virus.
 31. The immunogenic composition of claim29, wherein the second immunogenic peptide elicits a T-helper cellmediated response.
 32. An immunogenic composition comprising the peptideof claim 26 and a second immunogenic peptide conjugated to form aheteropolymer.
 33. An immunogenic composition comprising the peptide ofclaim 26 and a pharmaceutically acceptable lipid-containing carrier. 34.The immunogenic composition of claim 33, wherein the lipid-containingcarrier enhances a human T-lymphocyte response.
 35. The immunogeniccomposition of claim 33, wherein the lipid-containing carrier comprisesa lipoprotein.
 36. The immunogenic composition of claim 33, wherein thelipid-containing carrier comprises a liposome.
 37. The peptide of claim26, which is expressed by a DNA construct comprising a transcriptionalpromotor, a DNA sequence encoding said peptide, and a transcriptionterminator, each operably linked for expression of said peptide.
 38. Apeptide comprising from eight to thirteen amino acids and having atleast seven contiguous amino acids of a corresponding portion of HBpolhaving the sequence:

               (HBpol.sub.816-824) (SEQ ID No. 5)    Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu

wherein said peptide binds with an appropriate HLA molecule to form acomplex recognized by cytotoxic T cells which T cells recognize a nativeHBV antigen.
 39. The peptide of claim 38, which comprises from nine toeleven amino acids.
 40. The peptide of claim 38, which comprises:

         (HBpol.sub.816-824) (SEQ ID No. 5)    Phe-Leu-Leu-Ser-Leu-Gly-Ile-His-Leu.


41. An immunogenic composition comprising the peptide of claim 38 and asecond immunogenic peptide.
 42. The immunogenic composition of claim 41,wherein the second immunogenic peptide elicits an immune responsespecific for hepatitis B virus.
 43. The immunogenic composition of claim41, wherein the second immunogenic peptide elicits a T-helper cellmediated response.
 44. An immunogenic composition comprising the peptideof claim 38 and a second immunogenic peptide conjugated to form aheteropolymer.
 45. An immunogenic composition comprising the peptide ofclaim 38 and a pharmaceutically acceptable lipid-containing carrier. 46.The immunogenic composition of claim 45, wherein the lipid-containingcarrier enhances a human T-lymphocyte response.
 47. The immunogeniccomposition of claim 45, wherein the lipid-containing carrier comprisesa lipoprotein.
 48. The immunogenic composition of claim 45, wherein thelipid-containing carrier comprises a liposome.
 49. The peptide of claim48, which is expressed by a DNA construct comprising a transcriptionalpromotor, a DNA sequence encoding said peptide, and a transcriptionterminator, each operably linked for expression of said peptide.